Oxidative stress and inflammation play a crucial role in the pathogenesis of acute lung injury (ALI). Previously, pentapeptide bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was reported to possess significant antioxidant activity and inhibit lipopolysaccharides (LPS)-induced NF-κB activation in vitro, whereas little is known about its effects in vivo. In this study, we explored the effects of BP5 on endotoxemia-induced ALI in mice and the underlying molecular mechanisms. Our studies revealed that BP5 markedly improved survival and effectively alleviated lung injury by reducing overoxidation and excessive inflammatory response in endotoxemia mice. In LPS-stimulated mouse primary macrophages and RAW 264.7 cells, BP5 also exhibited antioxidant and anti-inflammatory properties by enhancing Nrf2 activation. Importantly, these beneficial effects were abolished by Nrf2 knockdown. To further elucidate the underlying mechanisms, we performed localized surface plasmon resonance (LSPR) assays, molecular docking, together with cell-based studies, and found that BP5 inhibited the Keap1-Nrf2 interaction to promote Nrf2 nuclear translocation and activation. Moreover, BP5-induced Nrf2 activation was shown to be accompanied by an increase in the phosphorylation of Akt (at Ser473) and GSK3β (at Ser9), and a decrease in Fyn nuclear accumulation both in vitro and in vivo. Pharmacologically inhibiting phosphorylation of Akt and GSK3β obviously enhanced Fyn nuclear accumulation in RAW 264.7 cells, which partially attenuated the promoting effect of BP5 on Nrf2 nuclear accumulation and activation. Furthermore, In Nrf2 mice, the protective effects of BP5 on the endotoxemia-induced ALI in WT mice were largely vanished. Our findings indicated that BP5 effectively protected endotoxemia-induced ALI against oxidative stress and inflammatory response, which are largely dependent on activation of the Nrf2 pathway. Underlying mechanisms include dual regulation of the Keap-Nrf2 interaction and the Akt (Ser473)/GSK3β (Ser9)/Fyn pathway.